Project Summary Adult-onset cardiomyopathies and heart failure are significant health and economic burdens throughout the world, with prevalence projected to increase by 46% by 2030. However, there is a paucity of animal models in which to test the etiologies and possible interventions in adult heart failure. This project has created two novel models of adult-onset cardiomyopathy and heart failure in zebrafish. The first model utilizes intersectional double transgenics to exclusively label Neural Crest derived Cardiomyocytes (NC-Cms), a small group of cardiomyocytes that are stereotypically positioned in ventricles, are required for normal patterning of trabeculae, and persist throughout adult life. Our double transgenics allow exclusive ablation of NC-Cm lineages at any stage of life; ablation during embryogenesis results in adult-onset cardiomyopathy and heart failure in exercise stress tests. The Notch pathway ligand jag2b is enriched in NC-Cms. Notch signaling is activated in adjacent mesoderm-derived cardiomyocytes, not in NC-Cms. These important molecular distinctions between cardiomyocyte lineages led to the second model, genetic mutants of jag2b, which have both embryonic trabeculation patterning defects and adult-onset cardiomyopathy. In contrast to zebrafish, adult mammals have evolutionarily lost the ability to regenerate hearts upon injury and apparently lack NC-Cms, lost either evolutionarily or developmentally. Synergistic analysis of these evolutionary distinctions between zebrafish and mammals will be informative. In response to ventricle resection, adult zebrafish lacking NC-Cms fail to regenerate normal hearts, implicating NC-Cms and their gene regulatory networks (GRNs) in adult heart regeneration. A multifaceted series of transgenic experiments will test the regeneration requirements of embryonically-derived NC-Cm lineages and de novo activation of NC-Cm GRNs in resected hearts. These results might lead to pathway interventions that can rescue the ability to regenerate in adult mammalian hearts. The proposed research uses two novel zebrafish models and a wide variety of tools, including lineage and temporally regulated transgenics, mutants, cardiac physiological, cutting-edge imaging and molecular approaches to understand the etiologies of adult-onset cardiomyopathy, heart failure and cardiac regeneration in zebrafish. This research will discover developmental mechanisms and GRNs that make NC-Cms distinct from their neighboring ventricle cardiomyocytes, and that are required to prevent adult-onset cardiomyopathy and facilitate adult heart regeneration. Our goals are to position these zebrafish models to test pharmacological and other interventions in order to contribute to our understanding and treatments of human heart diseases.